problems about myosin motility assay - (Jan/27/2008 )

hi everybody,i was trying the motility assay of myosin IX for nearly a year. but it really frustrated me that no moving was detected. I used the buffer exactly what others used in their papers, in which myosin moved quite well. I don’t know what happens. Does anyone have any ideas about the improvement? many thanks.

-lical208-

maybe your water quality is different from that used in the literature.

myosin is very sensitive to the presence or absence of ions.

-mdfenko-

QUOTE (mdfenko @ Jan 28 2008, 02:58 PM)

maybe your water quality is different from that used in the literature.

myosin is very sensitive to the presence or absence of ions.

dear mdfenko,

thank you very much for your reply. your suggestion always helps.

i know you've got a lot of knowledge of myosins. have you ever worked with myosin 9? what kind of buffer you may suggest for the storage of the myosin? I tried the buffer suggested in the literature, but it not worked well for myosin 9. the moysins were degraded completely within a month. many thanks.

-lical208-

QUOTE (lical208 @ Jan 30 2008, 05:33 AM)

i know you've got a lot of knowledge of myosins. have you ever worked with myosin 9? what kind of buffer you may suggest for the storage of the myosin? I tried the buffer suggested in the literature, but it not worked well for myosin 9. the moysins were degraded completely within a month. many you might thanks.

when i worked with myosin they were just starting to number them. we identified them by tissue type (skeletal muscle, smooth muscle, brain) and/or organism (chicken, rabbit, dog, bovine, human, slime mold (in the literature)). so, i haven't worked with myosin 9 (to my knowledge).

are you working with the same organism, myosin type as the paper you are following? you may need to optimize the buffer for yours.

is there any other literature you can follow for your myosin? to get clues for optimization.

is your myosin degrading or denaturing?

if it is degrading, is it purified or crude? you may need to add protease inhibitors.

if it is denaturing then you may need to modify your storage conditions. we store myosin at -20C after adding glycerol to 50% to the soluble protein (high salt, buffered, 0.5-2mM dtt before addition of glycerol). we precipitate by diluting then resuspend in high salt when we need to use some. we store at these conditions for years without significant loss.

storage at 4C is only okay for a couple of days before the solution "goes bad" (and starts to smell like used socks).

i hope some of this helps.

-mdfenko-

QUOTE (mdfenko @ Jan 30 2008, 07:50 AM)

when i worked with myosin they were just starting to number them. we identified them by tissue type (skeletal muscle, smooth muscle, brain) and/or organism (chicken, rabbit, dog, bovine, human, slime mold (in the literature)). so, i haven't worked with myosin 9 (to my knowledge).

are you working with the same organism, myosin type as the paper you are following? you may need to optimize the buffer for yours.

is there any other literature you can follow for your myosin? to get clues for optimization.

is your myosin degrading or denaturing?

if it is degrading, is it purified or crude? you may need to add protease inhibitors.

if it is denaturing then you may need to modify your storage conditions. we store myosin at -20C after adding glycerol to 50% to the soluble protein (high salt, buffered, 0.5-2mM dtt before addition of glycerol). we precipitate by diluting then resuspend in high salt when we need to use some. we store at these conditions for years without significant loss.

storage at 4C is only okay for a couple of days before the solution "goes bad" (and starts to smell like used socks).

i hope some of this helps.

dear mdfenko,

thank you very much. great ideas. i will check the things you mentioned.

but i am still surprised that you store your myosin at -20C, because my boss told that will kill the myosin. and i know some groups turn to store their myosins at -80C. have you also tried that?

-lical208-

QUOTE (lical208 @ Jan 30 2008, 01:19 PM)

but i am still surprised that you store your myosin at -20C, because my boss told that will kill the myosin. and i know some groups turn to store their myosins at -80C. have you also tried that?

no, we haven't tried storage at -80C.

we use -20C and prevent freezing by adding glycerol to 50%.

the myosin remains viable for several years (i have used >10yr with good result).

mind you, some preps last longer than others (probably depends on how "clean" they are). after precipitation and resuspension, i always check atpase activity (ca, mg and edta) before using it.